APEX-Ribo-Seq reveals that local translation occurs at 14 distinct subcellular compartments with over 3000 genes translated in compartment-specific patterns, regulated by diverse mechanisms including RNA-binding protein-mediated sequence recognition and onsite translational control that vary across organelles, with dynamic modulation during cell cycle progression and stress responses, and distinct translation programs in neuronal dendrites, axons, and presynapses.

Local translation at specific subcellular regions is proposed to define appropriate protein destinations and functions. However, our understanding of local translation is still far from complete, due to a lack of versatile analysis tools. Here, we developed a new method, termed “APEX-Ribo-Seq”, that integrates ribosome profiling (Ribo-Seq) with APEX2-based proximity labeling. We mapped local translation at 14 distinct organelles and identified over 3000 genes that are locally translated. Interpretable machine learning methods based on RNA language models systematically uncovered RNA-binding proteins with key roles in compartment-specific translation. In addition, we revealed slow translation kinetics associated with cotranslational membrane insertion of nascent chains. APEX-Ribo-Seq also captured cell cycle-regulated dynamics at the centrosome and stress-regulated programs at stress granules and autophagosomes. We also demonstrated the utility of APEX-Ribo-Seq in primary neurons, revealing compartment-specific translatomes in dendrites, axons, and presynapses. Our results provide a comprehensive view of the spatiotemporal translatome and its regulation.